New Characterization of Multi-Drug Resistance of Streptococcus suis and Biofilm Formation from Swine in Heilongjiang Province of China

The aim of this study was to investigate the antimicrobial resistance profiles and genotypes of Streptococcus suis in Heilongjiang Province, China. A total of 29 S. suis were isolated from 332 samples collected from 6 pig farms. The results showed that serotypes 2, 4 and 9 were prevalent, and all the clinical isolates were resistant to at least two antibacterial drugs. The most resisted drugs were macrolides, and the least resisted drugs were fluoroquinolones. Resistant genes ermB and aph (3′)-IIIa were highly distributed among the isolates, with the detection rates of 79.31% and 75.86%. The formation of biofilm could be observed in all the isolated S. suis, among which D-1, LL-1 and LL-3 strains formed stronger biofilm structure than other strains. The results indicate that S. suis in Heilongjiang Province presents a multi-drug resistance to commonly used antimicrobial drugs, which was caused by the same target gene, the dissemination of drug resistance genes, and bacterial biofilm.


Introduction
Streptococcus suis (S. suis), as an important zoonotic pathogen, can cause several symptoms, such as meningitis, arthritis, pneumonia, endocarditis, and septicaemia in pigs and humans [1]. S. suis can be divided into 35 serotypes based on S. suis capsular antigens [2], and serotypes 2, 4, 7 and 9 are frequently isolated from both healthy and diseased animals [3,4]. In order to control the S. suis infections, massive amounts of antimicrobials are used in the swine industry, which leads to the emergence of S. suis drug resistance strains worldwide. In Thailand, 99.3% of S. suis isolated from humans and pigs had resistance to at least one antibiotic drug [5]. In Korea, 95.6% of S. suis isolated from pigs were resistant to clindamycin, tilmicosin, tylosin, oxytetracycline, and more classes of antimicrobials [3]. In addition, most isolated clinical S. suis were resistant to clindamycin, tetracycline, and erythromycin in China from 2017 to 2019 [6]. All of the isolates were resistant to three or more categories of antimicrobial. Therefore, it is essential to monitor antimicrobial susceptibility of S. suis.
The clinically isolated S. suis often showed cross resistance between macrolides, lincosamides, aminoglycosides, chloramphenicol, β-lactams, fluoroquinolones, and tetracyclines [7][8][9]. Especially, high levels of resistance to tetracycline (TET) and macrolides In the study, 29 isolates of S. suis with multiple drug resistance features were collected from 332 clinical samples of pig farms in Heilongjiang Province of China from 2017 to 2018. Multi-drug resistance related factors were analyzed, including serotype, drug resistant phenotypes, drug resistance genes, and biofilm formation ability. Our findings explain some of the reasons for the development of multi-drug resistance by S. suis, providing a theoretical basis to understand, prevent, control, and reduce the occurrence of multiple drug resistance in S. suis as well as inhibit their biofilms formation ability.

Isolation and Identification of S. suis
Isolates were collected from pigs with symptoms of clinical mastitis across different regions in Heilongjiang Province (Figure 1). qPCR detection results showed that gdh of S. suis were positive. The Gram staining of S. suis appeared paired or short-chain. Of the 332 samples tested, 29 (8.7%) isolates were further characterized into various serotypes using cps genes (Figure 2). In the Harbin-1 farm, six S. suis strains were isolated, in which four isolates belonged to serotype 4, and two isolates belonged to serotype 2. In the Harbin-2 farm, three S. suis strains were isolated, in which one isolate belonged to serotype 2, one isolate belonged to serotype 9, and one isolate was unidentified. In the Harbin-3 farm, nine S. suis strains were isolated, all of which belonged to serotype 4. In the Suihua farm, one S. suis strain was isolated and unidentified. In the Daqing farm, three S. suis strains were isolated, in which two isolates belonged to serotype 4, and one isolate was unidentified. In the Qiqihar farm, seven S. suis strains were isolated, all of which belonged to serotype 2. Taken together, 10 S. suis strains of serotype 2 (34.48%), 15 S. suis strains of serotype 4 (51.72%), 1 S. suis strain of serotype 9 (3.45%), and 3 unidentified S. suis strains (10.35%) were isolated (Supplementary Table S1).

Biofilm Formation Analysis
Biofilm formation ability was observed in all of the isolated S. suis strains. According to the OD values, biofilm formation ability could be divided into three groups: strong, medium, and weak. Among these strains, clinical isolates, such as D-1, LL-1, and LL-3 The resistance genes of ermA, ermB, ermTR, mefA, mefE, msrD belong to macrolides, the aph (3 )-IIIa gene belongs to aminoglycosides, the lnu(B) gene belongs to lincosamides, the tetO and tetM genes belong to tetracyclines.

Discussion
S. suis is one of the leading causes of infectious diseases in pig farms globally. It is also considered as an emerging zoonotic agent for humans exposed to sick pigs or their by-products, with potential significant impact on public health. So far, the control or treat-

Discussion
S. suis is one of the leading causes of infectious diseases in pig farms globally. It is also considered as an emerging zoonotic agent for humans exposed to sick pigs or their by-products, with potential significant impact on public health. So far, the control or treatment of S. suis infections depends almost entirely on the use of antimicrobials. In the case of treating S. suis, antibiotic resistance is a common phenomenon, and there is an increasing trend of multi-drug resistance. An important approach is to focus on how to mitigate bacterial resistance. In order to promote the rational use of drugs, this study investigated the relationship of drug resistance and biofilm formation using a population of twenty-nine S. suis strains isolated from Heilongjiang Province.
S. suis serotype 2 is the main serotype causing pig and human infections in China [4]. However, in recent years, S. suis serotypes 4 and 9 have been frequently isolated from Chinese pig farms [30], and the isolation rate has gradually increased. Serotype 9 has been frequently isolated from diseased farm animals in intensively-reared commercial pig breeds [31], while serotype 4 has a high isolation rate from healthy pigs in China [32]. In some places in China, S. suis serotype 4 has become a dominant serotype [3,33]. Our present data identified at least four serotypes during the experimental period (June 2017 to August 2018) in Heilongjiang Province. This study confirmed that serotypes 2, 4, and 9 were prevalent in Heilongjiang. A total of 10 strains of S. suis type 2, 15 strains of S. suis type 4, and 1 strain of S. suis type 9 were identified; the ratios were 34.48%, 51.72%, and 3.45%, respectively. Obviously, S. suis serotype 4 was the main serotype in Heilongjiang during the investigation period. Our study found that strains GJ1-2, S-1, and S-2, isolated from the joints and abdomen of diseased pigs, belong to serotype 4, and exhibit strong multidrug resistance. Simultaneously, they have strong abilities of biofilm formation, suggesting that infections caused by serotype 4 S. suis are increasing. Although there has been a study reporting that S. suis serotype 2 is by far the most common isolated serotype related to disease [34], the increasing emergence of serotype 4 in infected pigs deserves more attention.
The massive use of antibiotics in the livestock industry contributes to the emergence of antibiotic resistance [7]. In China, the average usage of veterinary antibiotics has reached approximately 6000 tons annually, and most of them are used as feed additives, including tetracyclines and macrolides, among others [35]. In addition, lincosamides, macrolides, aminoglycosides, chloramphenicol, and β-lactams are also widely used in the treatment of S. suis [5,36]. In order to know the current drug resistance of S. suis in Heilongjiang Province, we tested the drug resistance phenotype and resistance gene of the 29 S. suis isolates. The detection results of the MICs of S. suis showed that the 29 clinical isolates were resistant to a minimum of two antimicrobial agents and a maximum of eight antimicrobial agents. Resistance to fluoroquinolones was the lowest, and the most resisted drugs were macrolides. Fluoroquinolone was relatively sensitive due to the reduction in the usage of fluoroquinolones in pig farms (a practice recently posed in China), while the massive use of macrolides, lincosamides, and tetracyclines could explain the serious drug resistant phenotype in this study.
The study indicated that all the isolated S. suis were 100% resistant to macrolides, which have been widely reported as a commonly used antibiotic in pig farms [37]. Drug resistant bacteria exhibit four drug resistance mechanisms, including target modification, active efflux, mutations in ribosomal L4 and L22, and production of inactivated enzymes [38]. Both erm and mef are acquired resistance genes. They are located in the transposon or carried by the plasmid, and can be transmitted between bacteria [39]. In a previous study, erythromycin and lincomycin developed cross-resistance due to modification of the same drug target site, which has been confirmed in their clinical isolates [40]. In our study, it was observed that 79.31% of the ermB genes detected in tylosin-resistant strains isolated from six farms were also resistant to lincomycin. This may be one of the reasons for the resistance to macrolides and lincomycin. Many drug resistance genes are located on integrative conjugative element (ICE), which can be passed through horizontal gene transfer to a new host cell, thus presenting a serious challenge to the pig industry. Transferable 89K-subtype ICEs, carrying ermBand tetO genes, can disseminate tetracycline and macrolide resistance genes in different swine and bovine farms of China [41]. Although not determined in this study, ICEs may lead to the resistance of the strains to macrolides and tetracycline in our research.
It is well known that biofilm formation by pathogens can significantly increase their tolerance to antibiotics [42]. Interestingly, S. suis tend to increase biofilm formation once exposed to sub-MICs of commonly used antimicrobials, including amoxicillin, enrofloxacin, oxytetracycline, and lincomycin. However, macrolides could inhibit the formation of S. suis biofilm in sub-MIC [43,44]. It is likely that some bacterial cells in biofilm are exposed to antibiotics of sub-MIC levels during antimicrobial chemotherapy, due to falling concentrations by dilution, or diffusion gradients for antibiotics in biofilm. Antibiotics of sub-MICs can provide a selective pressure, under which bacteria tend to mutate and become resistant to other antibiotics [24,42]. In our study, the reason for the stronger biofilm formation ability of LL-1, LL-3, and D-1 could be the long term administration of multi-antimicrobials in the pig farms in Qiqihar, which leads to biofilm formation. It has been reported that growing S. suis in the presence of sub-MIC of norfloxacin, doxycycline, and gentamicin is associated with increased biofilm formation ability, which promotes antibiotic tolerance [45]. Antimicrobials at sub-MIC can induce the production of extracellular polymeric substance (EPS) and further promote the resistance of biofilm. In this study, AH94-2 and AH94-4 were the same serotype S. suis isolated from the same farm (Harbin-1). Although the MIC of AH94-2 and AH94-4 to lincomycin were at the resistance level, and the gene lnu(B) was amplified, they showed different sensitivities to β-lactams. AH94-2 strain is more resistant to β-lactams than strain AH94-4. Meanwhile, isolates LL-3 and LQ-5 were obtained from a farm which used lincomycin, yet LL-3 showed stronger resistance to β-lactam drugs than LQ-5. We found that AH 94-2 and LL-3 exhibited stronger biofilm formation capacity than AH94-4 and LQ-5, which could reduce sensitivity to β-lactam antibiotics. That may explain the reason why AH 94-2 and LL-3 were more resistant than AH 94-4 and LQ-5, respectively. Another study also reported that biofilm-grown S. suis cells were much more resistant to penicillin G than planktonic cells [46]. Grenier also reported that the biofilm-grown S. suis was significantly more resistant to both penicillin G and ampicillin than planktonic cells [47]. It is possible that the EPS matrix forms a physical protective barrier for bacteria, which can prevent antimicrobials from penetrating into bacteria and ensure their normal growth [48]. In addition, the antibiotic degrading enzymes could be accumulated in the process of antibiotic penetration, which could inactivate the antibiotics and protect the bacteria within the biofilm [49]. Moreover, related resistance genes could not be amplified in macrolides resistant strain 2-5, aminoglycoside resistant strains B9-1, AY18-2, GJ1-2, and LL-1, and tetracycline resistant strains LL-1, G-3, DZ001-2, ZL695-2, Y10-2, AY18-2, and AHD-110-6-2, which indicates that the enhancement of biofilm formation may be related to the sensitivity of antimicrobial drugs. In addition, there is another possibility: that the PCR system was not able to detect all drug resistance genes. Notably, strain LL-1 did not contain resistance genes to tetracycline or aminoglycoside, yet exhibited resistance to both antimicrobials, which might be related to the strong biofilm formation ability. Biofilm grown cells may induce different physiological characteristics than the same isolate grown as colonies on the petri plate, and may have slightly different drug resistance phenotypes. Related studies have revealed that biofilm formation in bacteria could lead to the restriction of drug transport, which is the phase of positively charged antibiotics and a negatively charged EPS matrix [45]. Besides the EPS matrix and peptidoglycan, there are also other cell membrane components that could block the penetration of antibiotics. Bacteria could acquire more time from the delay of antibiotic penetration to achieve adaptive stress response [50]. The multi-speciation and heterogeneity of the bacterial community within the biofilm could cause a high probability of gene mutation, which will change the original gene structure and produce resistance via reducing or preventing binding of antibiotics to the target protein [29]. Interestingly, strain G3 contained resistance genes to aminogly-coside, yet did not exhibit resistance to aminoglycoside. It is possible that biofilm grown S. suis develops tolerance to antimicrobials, which enables S. suis to survive the MIC. One of the reasons for the development of bacteria tolerance is the slow growth of bacteria after biofilm formation, which causes the bacteria to enter into a growth quiescent period, thus reducing the susceptibility of biofilm to antibiotics [28]. Fridman et al. also reported that Escherichia coli cells spontaneously exhibited tolerance after repeated treatments of ampicillin [51]. Biofilm formation could provide bacteria with protection against host defense systems or antimicrobial agents, which leads to multidrug resistance and chronic infection of bacteria. Therefore, the biofilm formation is an important reason for the drug resistance of S. suis infections.
In conclusion, 29 clinical S. suis strains were isolated, and serotypes 2, 4, and 9 of S. suis were identified to be prevalent in Heilongjiang Province in this study. Among the isolated strains, the resistance rate of fluoroquinolones was the lowest, while the resistance rate of macrolides was 100%, indicating the serious situation of multi-drug resistance of S. suis in Heilongjiang Province. Resistant genes ermB and aph (3 )-IIIa were highly distributed among the 29 isolates. The inconsistency of the resistance genotypes and phenotypes of S. suis was due to the formation of biofilm, or could have been because the PCR/primer systems used in this study did not detect some drug resistant genes. In addition, this study did not measure drug resistance from biofilm grown isolates, only from colonies on petri plates. The physiological difference in growth conditions could also provide a reason for inconsistency in genotype and phenotype. The study provides us with detailed information on the prevalence and antimicrobial susceptibilities of S. suis in Heilongjiang Province from 2017 to 2018, which will enhance our knowledge in understanding how to prevent and control the problem of multi-drug resistance caused by S. suis in pig farms.

Samples
332 swabs were collected from 332 pigs of six swine farms in different regions of Heilongjiang Province (one sample/pig), during June 2017 to August 2018. The pigs were housed at the six farms under the same housing, feed, and environmental conditions. The room temperature is 22 • C, the room humidity is 55%, and the pigs were fed three times a day. The majority of the isolates were obtained from the nasal mucosa of asymptomatic pigs and others were obtained from the lungs, joints and abdomens of diseased pigs. Samples collected in Daqing, Qiqihar, and Harbin-3 are from animals that were treated with some antibiotics to cure the disease, while the samples from the other three farms are from animals that did not receive any antibiotic therapy (Supplementary Table S3). Of the available farm data, six farms accounted for twenty-nine isolates ( Table 2). In the Harbin-1 farm, 40 samples were collected and 6 S. suis were isolated. In the Harbin-2 farm, 26 samples were collected and 3 S. suis were isolated. In the Harbin-3 farm, 44 samples were collected and 9 S. suis were isolated. In the Suihua farm, 4 samples were collected and 1 S. suis was isolated. In the Daqing farm, 40 samples were collected and 3 S. suis were isolated. In the Qiqihar farm, 178 samples were collected and 7 S. suis were isolated. These samples were approved and conducted in accordance with the guidelines of the Animal Welfare and Research Ethics Committee of Northeast Agricultural University. Welfare-related assessments and interventions that were carried out prior to, during, and after the experiment under the strict guidance of the committee.

S. suis Isolation and Serotypes Identification
Swabs were used to take nasal mucosa or fresh tissues from the pigs. Samples were plated onto Sheep Blood Agar (SBA) (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA), and incubated at 37 • C for 24 h to 48 h. Isolates that showed alpha hemolysis on SBA were suspected to be S. suis. All suspected S. suis strains were further identified by classical biochemical methods, such as Gram staining (Solarbio, Beijing, China). Bacterial genome extraction (Takara, Dalian, China) was performed according to the manufacturer's instructions. The amplification and sequencing of glutamate dehydrogenase (gdh) genes were conducted to further molecularly identify the suspected isolates [52]. The S. suis serotype 2 strain ATCC 700794 was used as a positive control for PCR amplifications. In previous research, 33 serotypes of S. suis have been identified based on antigenic differences in the capsular polysaccharide. Serotypes 2, 4, 7, and 9 are common epidemic strains; serotypes of the 29 S. suis isolates were determined by PCR as previously described [53] (Supplementary Table S4).

Biofilm Formation
We used a 96-well plate to examine the formation of bacterial biofilm. The plates for biofilm cultivation should be sterile, flat-bottomed, 96-well polystyrene tissue culture treated microtiter plates with a lid. The S. suis strains were subcultured on Todd Hewitt Broth (THB) agar plates under sterile conditions for purification separation. A single colony was selected from the THB agar plates and inoculated in sterile THB liquid test tubes. The incubation was seeded in three wells using 200 µL of each sample. Negative control wells contained broth only. The test for each strain was repeated three times. After incubation at 37 • C for 72 h without shaking, the medium was removed by aspiration and the wells were washed twice with distilled water. The remaining attached bacteria were fixed with 200 µL of 99% methanol (Guoyao Ltd., China) per well, and the plates were emptied after 5 min and left to dry. Then, the plates were stained with 200 µL of 2% crystal violet (Guoyao Ltd., Shanghai, China) per well for another 5 min. The excess stain was rinsed off by placing the plate under running tap water. After the plates were air dried, the dye bound to the adherent cells was resolubilized with 200 µL of 33% (v/v) glacial acetic acid (Guoyao Ltd., China) per well. The amount of released stain was quantified by measuring the absorbance at 595 nm with a microplate reader (DG5033A, Huadong Ltd., Nanjing, China) [55]. The experiments were performed in triplicate, and the mean values of three measurements were reported.

Statistical Analysis
Statistical analysis of in vitro experiments was performed using GraphPad software. An unpaired t test was performed on qRT-PCR results. Values of p < 0.05 were considered significant, and all data were expressed as mean ± SD. *** p < 0.001; ** p < 0.01; * p < 0.05. In order to calculate the ability of biofilm formation accurately, the cut-off value (ODc) was required to be established to separate biofilm producing strains from non-biofilmproducing strains. The cut-off value (ODc) was defined as five standard deviations (SD) above the mean OD of the negative control. ODc = average OD of negative control + (5 × SD of negative control). Final OD value of a tested strain is expressed as average OD value of the strain reduced by ODc value (OD = average OD of a strain-ODc). ODc value is calculated for each microtiter plate separately. If a negative value is obtained, it should be presented as zero, while any positive value indicates biofilm production. Biofilm formation ability was classified according to the values of the ODs obtained: OD ≤ ODc, non-adherent; ODc < OD ≤ 2 × ODc, weak biofilm formation; 2 × ODc < OD ≤ 4 × ODc, moderate biofilm formation; and 4 × ODc < OD, strong biofilm formation [56,57]. Controls included THB medium alone (negative) and culture with S. suis ATCC 700794 (positive).

Conclusions
In this study, 29 clinical S. suis strains were isolated, and serotypes 2, 4 and 9 of S. suis were identified to be prevalent in Heilongjiang Province. Among the isolated strains, the resistance rate of fluoroquinolones was the lowest, while the resistance rate of macrolides was 100%, indicating the serious situation of multi-drug resistance of S. suis in Heilongjiang Province. In conclusion, the study provides us with detailed information on the prevalence and antimicrobial susceptibilities of S. suis in Heilongjiang Province from 2017 to 2018, which will enhance our knowledge in understanding how to prevent and control the problem of multi-drug resistance caused by S. suis in pig farms.